Subtilisin variants with improved characteristics

ABSTRACT

The present invention provides subtilisin variants with improved characteristics, including improved substrate affinity, catalytic activity, catalytic efficiency and stability under washing conditions as well as overall wash performance. The subtilisin variants are therefore useful additives in cleaning compositions.

FIELD OF THE INVENTION

[0001] The present invention provides subtilisin variants with improvedcharacteristics, including improved substrate affinity, catalyticactivity, catalytic efficiency and stability under washing conditions aswell as overall wash performance. The subtilisin variants are thereforeuseful additives in cleaning compositions.

BACKGROUND OF THE INVENTION

[0002] Proteases are enzymes that catalyze the hydrolysis of peptidebonds in a protein's backbone. Protease with low substrate specificityare required in order to degrade various protein soils to smallerfragments and, thereby, to support a detergent's action of removingstains like egg, grass, grain etc. from textiles and other materials.Subtilisins are used for this purpose for more than 30 years with largesuccess, mainly as additives in cleaning compositions, e.g. laundrydetergents. Consequently, detergent proteases account today forapproximately 30% of total worldwide enzyme production. A large numberof different subtilisins and subtilisin-like enzymes have been isolatedand cloned from several microorganisms including mainly bacillus species(e.g. Bacillus subtilis, Bacillus lentus, Bacillus licheniformis,Bacillus amyloliquefaciens), but also from other organisms (e.g.Tritirachium album, Thermoactinomyces vulgaris). However, only a fewsubtilisins have been used commercially as additives for cleaningcompositions, mainly due to the fact that the technical conditions underwhich the enzyme is used are far away from natural conditions. Forapplication in cleaning compositions subtilisins need to be stable andactive under the harsh conditions of the automatic washing procedure(medium to high temperature, alkaline pH, oxidizing conditions,surfactants, etc.). Consequently, a large number of studies have beendone on the design of subtilisins to fulfill these requirements.

[0003] Properties of a protease can be improved by introducing mutationsinto the amino acid sequence of a precursor, subtilisin at definedpositions. Such efforts have lead to better subtilisins as disclosed inseveral patent applications. For example, U.S. Pat. No. 5,346,823describes subtilisin derivatives which are modified by substitution ofmethionine, trypthophane, lysine or cysteine with alanine or serineresulting in improved stability under highly oxidizing conditions.

[0004] Analogously, a number of subtilisin variants have been obtainedthat are better suited for technical conditions such as highly alkalinepH, high temperature, high concentrations of detergents, etc. Accordingto U.S. Pat. No. 5,246,849 thermally stable serine proteases are createdby introducing cysteine at position 206. Subtilisins with enhancedthermal stability were obtained by modifications at the calcium-bindingsites at positions 131 and 172 (see U.S. Pat. No. 5,260,207). Subtilisinvariants showing alterations in characteristics as thermal and alkalinestability were described in U.S. Pat. No. 5,972,682. Besides ofstability, the utility of an enzyme is further characterized by itscatalytical behavior in its proposed technical field. Related parameterssuch as proteolytic activity, wash performance, were also alreadysubject of several patents. For example, single amino acid substitutionsat position 225 and at positions 224 as disclosed in U.S. Pat. No.5,155,033 and in U.S. Pat. No. 5,182,204, respectively, resulted in atleast 1.5 fold enhanced catalytic activity of the modified subtilisincompared to its precursor subtilisin.

[0005] Multi-mutations also can result in increased proteolytic activityas disclosed in EP-A-0451244 describing substitutions at position 123and 274. U.S. Pat. No. 6,287,841 discloses subtilisin mutants derivedfrom PB92 subtilisin or Subtilisin 309 by substitutions at combinationsof positions 60, 87, 97, 99, 102, 116, 117, 126, 127, 128, 130, 133,134, 154, 158, 159, 160, 164, 169, 175, 180, 182, 193, 197, 198, 203,211 and 216 corresponding to these precursor subtilisins, havingimproved wash performance or/and improved stability.

[0006] As another example, the subtilisins described in WO 91/00345 andEP-A-0405901, with change of the net electrostatic charge in favour toan isoelectric point lower or higher than that of the parentsubtilisins, resulting in subtilisins with improved adsorption to thesubstrate, i.e. protein soils on textile surfaces or other material tobe cleaned, encompass among other mutations a modification done atposition 54 with the glutamic acid being substituted by Thr, Tyr or Gly.WO 92/11348, as well as U.S. Pat. Nos. 5,631,217 and 5,482,849 disclosethe substitution at position 54 by aspartic acid.

[0007] U.S. Pat. No. 5,316,935 discloses modifications selected fromD60N, D97G, Q103R, G131D, S182G, S188P, D248A, D248L, D248N or T255, andoptionally E156G and/or N218S when the subtilisin already contains thesubstitution N181S resulting in modified subtilisins as catalysts withimproved stability and/or catalytic activity in organic solvents.

[0008] U.S. Pat. No. 4,760,025 describes subtilisin variants derivedfrom subtilisin naturally produced by Bacillus amyloliquefaciens thatare substituted at positions 32, 155, 104, 222, 166, 64, 33, 169, 189,217, 157 by any different amino acid. U.S. Pat. No. 5,700,676 alsopertains to improved subtilisin modified at selected positions bysubstitution of the amino acid with a different naturally occurringamino acid.

[0009] According to U.S. Pat. No. 6,197,567, mutant subtilisinsdistinguished from the parent subtilisins by different amino acids atone or more selected positions with the different amino acid being morepositive or negative than the amino acid in the corresponding positionin the parent subtilisin resulting in enzyme mutants exhibiting improvedwash performance.

[0010] WO 95/07991 describes subtilisin mutants with improved propertiesas decreased adsorption to and increased hydrolysis of an insolublesubstrate, wherein the modifications are defined substitutions in therange of positions 199 to 200, either at least at one position or at twopositions. For example, in case of a double substitution there are anumber of amino acids proposed substituting the tyrosine at position 217with the preferred amino acid being leucine. EP-A-0380362, whichpertains to the use of subtilisin mutants in chemical reaction in anon-native environment, discloses lysine or leucine as appropriatesubstituating amino acids at position 217, whereas WO 96/09396 describesa calcium-free thermostable subtilisin mutant comprising themodification Y217K, inter alia.

[0011] WO 01/07578 describes subtilisin-like protease variants havingdecreased immunogenicity evolved by the combination of definedsubstitutions at one or more positions in one or more epitope regions(ranging from 108 to 126 AA; 221-246 AA), at two or more positions inone or more epitope region (113-125 AA; 221-246 AA), at one or morepositions in two or more of the epitope regions (108-125 AA; 221-246 AA;70-84 AA) or at two or more positions in one or more of the epitoperegions (ranging from 103-126 AA, 220-246AA, 70-84 AA).

[0012] WO 95/30010 concerns variants with decreased adsorption to andincreased hydrolysis of an insoluble substrate and discloses definedamino acid substitutions in six loop regions of BPN′ subtilisin, withthe first loop region ranging from AAs 59 to 66, the second loop fromAAs 95 to 107, the third loop from AAs 126 to 133, the fourth loop fromAAs 154 to 167, the fifth loop from AAs 187 to 191, and the sixth loopfrom AAs 199 to 220 with the BPN′ variants having modified amino acidsequences comprising one or more substitutions in one or more of theloop regions (first to fifth) or modifications as described within thesixth loop region in combination with substitution(s) in one of theaforementioned loop regions.

[0013] WO 95/10615 describes subtilisin variants with altered propertiescompared to those of the precursor subtilisins having a substitution atposition 76 and further substitutions at selected positions (99, 101,103, 104, 107, 123, 27, 105, 109, 126, 128, 135, 156, 166, 195, 197,204, 206, 210, 217, 218, 222, 260, 265, 274) with the main substitutionbeing N76D.

[0014] WO 99/20770 pertains to subtilisin variants with a substitutionat position 103 in combination with one or more substitutions at aminoacid residues in the range from 1 to 275, with the main substitutionbeing defined as S103A.

[0015] The variants according to U.S. Pat. No. 6,312,936 comprisesubstitutions at position 103 and 236 and/or 245 and furthersubstitutions within the range from AAs 1 to 275 with the mainsubstitutions being S103A, Q236H, Q246L.

[0016] Although a lot of progress has been achieved in the field ofsubtilisin modifications, there is continuing interest in new proteasevariants with further enhanced characteristics. One major point ofcommon interest is the still enormous amount of protease—be itengineered or not—needed for the technical application in the cleaningprocess. Taking laundry detergents as an example, the concentration ofprotease needed for a sufficient removal of protein stains from textilesis between 1 and 5 mg per liter washing liquid. This leads to aworldwide annual consumption of more than 500.000 kg enzyme per year.Furthermore, the high protease concentration corresponds to an increasedchance of exposure to the potentially immunogenic protein and, thereby,to an increased probability of allergenic effects.

[0017] Therefore, the underlying problem of the present invention is toprovide subtilisin variants with improved characteristics, especiallysubtilisin variants that can be applied technically as additives incleaning compositions at significantly lower concentrations as comparedto known subtilisin variants. Further improved characteristics mayinclude, but are not limited to, higher catalytic rates underapplication conditions, higher substrate affinity under applicationconditions, faster removal of protein stains from the particular surfaceunder application conditions, improved stain removing ability at lowlaundering temperature, better wash performance, improved stability uponstorage, improved stability during use, and improved resistance toself-degradation under application conditions. The present inventionprovides subtilisin variants that are improved for at least one of thesecharacteristics. These subtilisins are useful additives in cleaningcompositions such as laundry detergents and other technical orindustrial applications.

SUMMARY OF THE INVENTION

[0018] The Present Invention Provides

[0019] (1) a subtilisin variant having improved characteristics undertechnical conditions such as a laundry detergent solution, in particulara modified subtilisin having at least one mutation in an amino acidsequence of a precursor subtilisin at a position numbered according tothe amino acid sequence of the mature subtilisin BPN′ shown in SEQ IDNO:1, wherein said at least one mutation is selected from (a) E54D orE54G, (b) Q103R or Q103K, (c) T133K, (d) E156K or E156A, and (e) Y217H,or mutations at the respective positions which are homologous to saidmutations (a) to (e), provided that in case of mutations E54D, Q103R,E156K or E156A the modified subtilisin has at least one more of saidmutations (a) to (e);

[0020] (2) a modified subtilisin as defined in (1) above which has atleast two of the following group of amino acid substitutions: E54D,Q103R, T133K, E156A, Y217H;

[0021] (3) a modified subtilisin as defined in (1) or (2) above which isselected from variants 1 to 50 defined hereinbelow;

[0022] (4) a DNA coding for the modified subtilisin as defined in (1) to(3) above;

[0023] (5) a vector comprising the DNA as defined in (4) above;

[0024] (6) a microorganism which comprises the DNA as defined in (4)above and/or the vector as defined in (5) above;

[0025] (7) a method for producing the modified subtilisin as defined in(1) to (3) above which comprises culturing a microorganism as defined in(6) above;

[0026] (8) a detergent composition comprising a modified subtilisin asdefined in (1) to (3) above; and

[0027] (9) a method for degrading proteinaceous material which comprisestreating the proteinaceous material with a modified subtilisin asdefined in (1) to (3) above.

SHORT DESCRIPTION OF THE FIGURES

[0028]FIG. 1 shows a plasmid map of the shuttle vector pBVP43-Sub.

[0029]FIG. 2 shows an alignment of several subtilisins of the genusBacillus, wherein Sub DY is subtilisin DY (Bacillus subtilis; SEQ IDNO:3), Sub BLI is subtilisin Carlsberg (Bacillus licheniformis; SEQ IDNO:4), Sub E is subtilisin E (Bacillus subtilis; SEQ ID NO:2), Sub BPNis subtilisin BPN′ (Bacillus amyloliquefaciens; SEQ ID NO:1) and Sub Savis subtilisin Savinase (Bacillus lentus; SEQ ID NO:5).

[0030]FIG. 3 shows “exemplarily” the improvement of subtilisin variantsrelatiy to the subtilisin E wild type over 4 rounds of directedevolution towards higher activity in laundry detergents.

[0031]FIG. 4 shows the protein degradation performance of subtilisinvariant 22 in comparison to wild type subtilisins. All enzymes wereapplied at concentrations of 2.5 mg/l.

[0032]FIG. 5: Determination of the amount of enzyme of variant 50, 1, 4or 22 compared to the subtilisin E wild type needed in order to achievea certain protein degradation.

[0033]FIG. 6 shows a Michaelis Menten plot of subtilisin variants 1, 4,22. All enzymes were applied at concentrations of 2.5 mg/l.

[0034]FIG. 7 shows a comparison of the protein degradation andsolubilization performance between a laundry detergent containingvariant 22 and a laundry detergent containing a commercially availablesubtilisin.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention provides subtilisin variants with improvedcharacteristics compared to those of the respective precursor subtilisinenzymes. These improved properties enable a better utility in cleaningcompositions.

[0036] In the context of this invention “subtilisins” are serineproteases with low substrate specificity and structural homology tosubtilisin E from Bacillus subtilis (Biochim. Biophys. Acta 1543 (2000)203-222). Serine proteases are characterized by the catalytic triadecomprising aspartate-histidine-serine, reading from the amino to thecarboxy terminus. Well characterized subtilisins derive from bacterialstrains, such as subtilisin E (Bacillus subtilis), subtilisin 309(Bacillus lentus), subtilisin BPN′ (Bacillus amyloliquefaciens) orsubtilisin Carlsberg (Bacillus licheniformis). Derived from differentspecies the amino sequences of naturally occuring subtilisins are notentirely homologous, but differ in amino acid substitutions, insertionsand deletions. For the purpose of this invention all positions mentionedin the context of subtilisin are equivalent to the numbering of themature form of the naturally occuring subtilisin BPN′ of Bacillusamyloliquefaciens.

[0037] The amino acids mentioned are abbreviated according to thefollowing list either in one- or in three-letter code. AbbreviationsAmino acid A Ala Alanin C Cys Cysteine D Asp Aspartic acid E GluGlutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I IleIsoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn AsparagineP Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T ThrThreonine V Val Valine W Trp Tryptophane Y Tyr Tyrosine

[0038] The present invention provides “variants” of subtilisins. A“variant” in accordance with the invention has an amino acid sequencenot found in nature, but is instead derived from the amino acid sequenceof a “precursor” enzyme by one or more amino acid substitutions (in thepresent invention also referred to as “mutation”).

[0039] According to the invention, “precursor” subtilisins can benaturally occuring subtilisins such as subtilisin E (Bacillus subtilisstrain 168; SEQ ID NO:2), subtilisin BPN′ (Bacillus amyloliquefaciens;SEQ ID NO:1), subtilisin DY (Bacillus subtilis strain DY; SEQ ID NO:3),subtilisin Carlsberg (Bacillus licheniformis; SEQ ID NO:4), subtilisinsavinase (Bacillus lentus; SEQ ID NO:5), subtilisin J (Bacillusstearothermophilus; SEQ ID NO:6), alkaline elastase YaB (Bacillus sip.strain YaB; SEQ ID NO:7) and the alkaline protease precursor proteinsshown in SEQ ID Nos:8 and 9, or a fragment or derivative thereof,preferably the precursor subtilisin is subtilisin E (SEQ ID NO:2) or afragment or derivative thereof, or is a recombinant or engineeredsubtilisin, but also engineered subtilisins having at least 70% homologyin its amino acid sequence to a naturally found subtilisin. Furthermore,recombinant subtilisins (being derived from a combination of amino acidsequences of two or more precursor subtilisins) can serve as precursorsubtilisins.

[0040] By introducing mutations in the amino acid sequence of aprecursor subtilisin the characteristics of the resulting subtilisin canbe improved. In the context of the present invention, thecharacteristics of the subtilisin include one, several, or all of thefollowing parameters “Km”, “catalytic activity”, “catalytic efficiency”and “wash performance”. The Km (or “Michaelis Menten constant”) and thecatalytic activity are kinetic parameters of an enzyme according to thedefinitions of Michaelis and Menten (see Fersht, A., Enzyme Structureand Mechanism, W. H. Freeman and Company, New York, 1995). The term Kmdescribes the affinity of the enzymes to its substrate. The “catalyticactivity” used herein describes the rate of conversion of the substrateunder defined conditions. In the context of this invention, kinetic dataare obtained by measuring the hydrolysis of substrates over time.Substrates were fluorescent labeled casein or bovine serum albuminmolecules. The degradation was measured over time using confocalfluorescence spectroscopy (see WO9416313). The term “wash performance”used in this invention describes the solubilization and degradation ofproteins bound to textiles by the investigated protease applied in adetergent without proteases and compared to the effect of this detergentalone.

[0041] In the context of this invention, the term “original amino acid”means the amino acid found in the subtilisin E wild type enzyme at theparticular position. It should be noted, that if another subtilisin isto be improved by introducing the amino acid substitutions disclosed inthe present invention, the wild type amino acid residue at theparticular position might differ from the “original amino acid”.Substitutions are usually described as follows: original amino acid;position; substituted amino acid.

[0042] The subtilisin variants generated by means of directed evolutionas described in the Experimental Section have surprisingly been found tohave improved characteristics as industrial enzymes such as detergentproteases.

[0043] The subtilisin variants according to embodiment (1) of thepresent invention comprise one or a combination of the particularmutations (a) to (e), or mutations at the the respective positions whichare homologous to said mutations (a) to (e).

[0044] In particular, the mutation

[0045] (a) E54D or E54G means that at position 54, glutamic acid isexchanged by an aspartic acid or a glycine;

[0046] (b) Q103R or Q103K means that at position 103, glutamine isexchanged by an arginine or a lysine;

[0047] (c) T133K means that at position 133, threonine is exchanged by alysine;

[0048] (d) E156K or E156A means that at position 156, glutamic acid isexchanged by a lysine or an alanine; and

[0049] (e) Y217H means that at position 217, tyrosine is exchanged by ahistidine. “Homologous mutations” according to the present invention aremutations which a person skilled in the art would consider homologous,i.e. equivalent. Thus if the mutated (the newly introduced) amino acidis e.g. a lipophilic amino acid, such as Leu, then other lipophilicamino acids such as Ile are also applicable.

[0050] In a preferred embodiment of the invention the subtilisincomprises at least two of the mutations (a) to (e) which includes thefollowing substitution combinations with the numbering of the substituedamio acid resides equivalent to the numbering of Bacillusamyloliquefaciens subtilisin (subtilisin BPN′).

[0051] E54D/Q103R, E54G/Q103R, E54D/Q103K, E54G/Q103K, E54D/T133K,E54G/T133K, E54D/E156K, E54G/E156K, E54D/E156A, E54G/E156A, E54D/Y217H,E54G/Y217H, Q103R/T133K, Q103R/E156K, Q103R/E156A, Q103R/Y217H,Q103K/T133K, Q103K/E156K, Q103K/E156A, Q103K/Y217H, T133K/E156K,T133K/E156A, T133K/Y217H, E156K/Y217H, E156A/Y217H, E54D/Q103R/T133K,E54G/Q103R/T133K, E54D/Q103R/E156K, E54G/Q103R/E156K, E54D/Q103R/E156A,E54G/Q103R/E156A, E54D/Q103R/Y217H, E54G/Q103R/Y217H, E54D/Q103K/T133K,E54G/Q103K/T133K, E54D/Q103K/E156K, E54G/Q103K/E156K, E54D/Q103K/E156A,E54G/Q103K/E156A, E54D/Q103K/Y217H, E54G/Q103K/Y217H, E54D/T133K/E156K,E54G/T133K/E156K, E54D/T133K/E156A, E54G/T133K/E156A, E54D/T133K/Y217H,E54G/T133K/Y217H, E54D/E156K/Y217H, E54G/E156K/Y217H, E54D/E156A/Y217H,E54G/E156A/Y217H, Q103R/T133K/E156K, Q103R/T133K/E156A,Q103R/T133K/Y217H, Q103R/E156K/Y217H, Q103R/E156A/Y217H,Q103K/T133K/E156K, Q103K/T133K/E156A, Q103K/T133K/Y217H,Q103K/E156K/Y217H, Q103K/E156A/Y217H, T133K/E156K/Y217H,T133K/E156A/Y217H, E54D/Q103R/T133K/E156K, E54G/Q103R/T133K/E156K,E54D/Q103R/T133K/E156A, E54G/Q103R/T133K/E156A, E54D/Q103R/T133K/Y217H,E54G/Q103R/T133K/Y217H, E54D/Q103R/E156K/Y217H, E54G/Q103R/E156K/Y217H,E54D/Q103R/E156A/Y217H, E54G/Q103R/E156A/Y217H, E54D/Q103K/T133K/E156K,E54G/Q103K/T133K/E156K, E54D/Q103K/T133K/E156A, E54G/Q103K/T133K/E156A,E54D/Q103K/T133K/Y217H, E54G/Q103K/T133K/Y217H, E54D/Q103K/E156K/Y217H,E54G/Q103K/E156K/Y217H, E54D/Q103K/E156A/Y217H, E54G/Q103K/E156A/Y217H,E54D/T133K/E156K/Y217H, E54G/T133K/E156K/Y217H, E54D/T133K/E156A/Y217H,E54G/T133K/E156A/Y217H, Q103R/T133K/E156K/Y217H,Q103R/T133K/E156A/Y217H, Q103K/T133K/E156K/Y217H,Q103K/T133K/E156A/Y217H, E54D/Q103R/T133K/E156K/Y217H,E54D/Q103R/T133K/E156A/Y217H, E54G/Q103R/T133K/E156K/Y217H,E54G/Q103R/T133K/E156A/Y217H, E54D/Q103K/T133K/E156K/Y217H,E54D/Q103K/T133K/E156A/Y217H, E54G/Q103K/T133K/E156K/Y217H,E54G/Q103K/T133K/E156A/Y217H.

[0052] More preferably the subtilisin of the invention comprises atleast three, even more preferably at least four and most preferably atleast five of said mutations (a) to (c).

[0053] The subtilisin of the invention with at least two modifications(a) to (e) includes:

[0054] (i) a subtilisin according to embodiment (1) which has a mutation(a), preferably the modification E54D, and has at least one (1),preferably at least two (2), more preferably at least three (3) and mostpreferably at least four (4) additional mutations selected from themutations (b) to (e) (particularly preferred is a subtilisin accordingto (i) above which has at least the additional modifications Q103R andE156A);

[0055] (ii) a subtilisin according embodiment (1) which has a mutation(b), preferably the modification Q103R, and has at least one (1),preferably at least two (2), more preferably at least three (3) and mostpreferably at least four (4) additional mutations selected from themutations (a) and (c) to (e) (particularly preferred is a subtilisindefined above, which has modifications Q103R and E156A and at least onefurther modification (a), (c) or (e), preferably the additional mutationT133K);

[0056] (iii) a subtilisin according to embodiment (1) which hasmodification (c) and has at least one (1), preferably at least two (2),more preferably at least three (3) and most preferably at least four (4)additional mutations selected from the mutations (a), (b), (d) and (e);

[0057] (iv) a subtilisin according to embodiment (1) which has amutation (d), preferably the modification E156A, and has at least one(1), preferably at least two (2), more preferably at least three (3) andmost preferably at least four (4) additional mutations selected from themutations (a) to (c) and (e); and

[0058] (v) a subtilisin according to embodiment (1) which has a mutation(e) and has at least one (1), preferably at least two (2), morepreferably at least three (3) and most preferably at least four (4)additional mutations selected from the mutations (a) to (d).

[0059] According to the embodiment (2) of the invention, the improvedsubtilisin variant comprises a combination of at least any two of thefollowing group of amino acid substitutions: E54D, Q103R, T133K, E156A,Y217H.

[0060] The subtilisin variants according to embodiments (1) and (2) ofthe present invention may comprise additional amino acid substitutionincluding substitutions at the following positions: 4, 5, 6, 7, 9, 14,15, 17, 18, 19, 20, 24, 25, 31, 38, 43, 45, 49, 50, 51, 54 modifieddifferently as compared to mutation (a), 56, 59, 61, 63, 78, 79, 89, 90,101, 103 modified differently as compared to mutation (b), 104, 111,118, 130, 133 modified differently as compared to mutation (c), 136,138, 145, 149, 150, 156 modified differently as compared to mutation(d), 157, 161, 164, 167, 173, 181, 183, 184, 185, 191, 197, 199, 204,206, 209, 217 modified differently as compared to mutation (e), 218,224, 228, 234, 235, 242, 243, 245, 247, 248, 249, 251, 252, 253, 259,263, 265, 271, 267, 269, 271, 272, 273 and 275.

[0061] Particularly preferred is that the additional mutation isselected from the following group of amino acid substitutions:

[0062] V4A, P5L, Y6N, Y6H, G7S, S9P, S9A, P14T, A15G, A15V, H17L, S18T,Q19H, G20D, S24T, N25D, I31L, S38T, N43S, N43I, N43T, R45S, S49G, F50I,V51I, N56S, Q59L, G61S, S63P, S78P, I79F, S89P, L90I, Y104N, I111V,I111N, N118Y, T130I, T130A, K136N, V138A, S145G, V149I, A150V, E156G,G157D, S161N, T164S, Y167F, N181S, S183N, N184Y, Q185H, S191G, D197G,M199V, S204T, Q206H, Q206L, L209H, N218I, N218T, T224A, A228T, I234N,L235I, L235P, T242A, N243S, N243H, N243T, Q245L, D248V, R247H, R249S,S252G, T253S, N259H, Y263C, Y263N, K265N, L267F, N269H, N269I, N269D,Q271H, Q271L, A272P, Q275L.

[0063] In particularly preferred embodiment (3) of the invention, theimproved subtilisin is one of the following variants comprisingparticular combinations of amino acid substitutions (with the numberingof the substituted amino acid resides equivalent to the numbering ofsubtilisin BPN′):

[0064] Variant 1:Y6N/I31L/S38T/F50I/Q103R,

[0065] variant 2: P14T/Q103R/T130I/Q185H/K265N,

[0066] variant 3: V511/Q103R/E156A,

[0067] variant 4: Q103R/V138A/A150V/E156A/Y217H/Q271H,

[0068] variant 5: S9P/R45S/Q103R/T130A/E156G/S183N/Y217H,

[0069] variant 6: G61S/S78P/Q103R/E156A/

[0070] variant 7: E54D/I79F/Q103R/E156G/Y217H/N243S,

[0071] variant 8: S78P/L90I/Q103R/E156A,

[0072] variant 9: Q103K/T130A/E156G/L209H,

[0073] variant 10:Q103K/E156A/T242A/N259H,

[0074] variant 11: N25D/Q103K/E156A,

[0075] variant 12: P14T/A15G/E54D/Q103K/T130A/E156G/S252G/Q271L,

[0076] variant 13: Q103K/T133K/N181S/S252G/Q271L,

[0077] varinat 14: N43I/G61S/S78P/Q103K/T130A/E156G/S204T

[0078] variant 15: N43S/E54D/Q103R/S145G/E156K/G157D/M199V/Y217H/D248V,

[0079] variant 16: S18T/N43S/Q103R/V138A/A150V/E156A/Y217H/Q271L,

[0080] variant 17: P14T/A15V/H17L/Q103R/T130A/E156A,

[0081] variant 18: Q103R/V138A/A150V/E156A/Y217H/Q271H,

[0082] variant 19: Q103R/N181S/S204T/Y217H/T224A/A228T,

[0083] variant 20: V4A/E54D/G61S/Q103R/E156A/S183N/Y217H,

[0084] variant 21: S9P/E54G/Q103R/I111V/E156A/Y217H,

[0085] variant 22: E54D/Q103R/T133K/E156A/Y217H,

[0086] variant 23: S24T/G61S/Q103R/E156A/S183N/Y217H/T253S,

[0087] variant 24: G20D/N43T/E54D/Q103R/T130A/E156A/S183N/Y217H/L235I,

[0088] variant 25: V4A/E54D/G61S/Q103R/S145G/E156A/S183N/Y217H,

[0089] variant 26: Q103R/V138A/A150V/E156A/Y217H/D248V/R249S,

[0090] variant 27: P14T/A15V/I31L/N43S/E54D/Q103R/E156A,

[0091] variant 28:Q19H/I31L/E54D/Q59L/S63P/Q103R/Y104N/T130A/E156G/S161N/S183N/N243H/L267F/N269H,

[0092] variant 29:E54D/Q103R/I111N/N118Y/T130A/E156A/Q185H/I234N/L235P/Q245L/N259H,

[0093] variant 30: E54D/Q103R/I111N/N118Y/T130A/E156A/Q185H,

[0094] variant 31: E54D/T130A/V149I/E156A/S183N/D197G/Q206H/N218I/N269I,

[0095] variant 32: E54D/Q103R/T133K/K136N/E156A/N269D,

[0096] variant 33: Y6N/P14T/A15V/I31L/N43S/E54D/Q103R/E156A,

[0097] variant 34: P14T/A15V/E54D/Q103R/E156A/Y167F/Y217H/N218T,

[0098] variant 35:P14T/A15V/I31L/E54D/Q103R/N118Y/E156A/S191G/N243T/Y263C/N269H,

[0099] variant 36: G7S/S9A/Q103R/T133K/E156A/Y217H/T224A/Y263N/A272P,

[0100] variant 37: E54D/Q103R/T133K/K136N/E156A/Y217H,

[0101] variant 38: S9P/Q103R/T133K/E156A/T164S/S183N/Y217H,

[0102] variant 39: E54D/Q103R/T133K/E156A/N184Y/Q206L/Y217H/K265N,

[0103] variant 40: P5L/Y6H/S49G/Q103R/T133K/E156A/Y217H,

[0104] variant 41: E54G/Q103R/I11V/V138A/A150V/E156A/Y217H/L235I,

[0105] variant 42:S89P/Q103R/E156A/Q271H,

[0106] variant 43: I31L/N43S/N56S/Q103R/V138A/A150V/E156A/Y217H/Q271H,

[0107] variant 44: P14T/A15V/E54D/Q103R/E156A/Y217H/Q271H,

[0108] variant 45: E54D/Q103R/T133K/E156A,

[0109] variant 46: E54D/Q103R/T133K,

[0110] variant 47: V4A/E54D/G61S/Q103R/S183N,

[0111] variant 48: S9P/E54D/Q103R/I111 V/S183N/D248V/R249S,

[0112] variant 49: N43S/E54D/Q103R/S145G/E156K/G157D/M199V/Y217H/D248V

[0113] variant 50: S145G/E156K/R247H

[0114] Among the variants 1 to 50 those derived from subtilisin E asshown in SEQ ID NOs:10 to 59 are particularly preferred.

[0115] The substitutions in the amino acid sequence of a precursorsubtilisin result in subtilisin variants that surprisingly show enhancedcharacteristics compared to the precursor subtilisin. The subtilisinvariants have distinguishing properties, such as improved catalyticactitvity as compared to the precursor subtilisins. The subtilisinvariants of interest in this invention have a lower Km and a highercatalytic activity in comparision to the precursorsubtilisin.Furthermore, the subtilisin variants of interest in this inventionsurprisingly show an improved wash perfomance in laundry detergent. Theimprovement of wash performance is connected with a better result inproteinaceous stain removal or otherwise stated less of the subtilisinvariant has to be added to obtain the same result as with the precursorsubtilisin. Furthermore, these variants show a better stability inpresence of typical ingredients of cleaning composition as oxidizingagents.

[0116] Moreover, the invention comprises also the use of the subtilisinvariants as defined herein before as additive in a cleaning composition.This cleaning composition may be used as laundry detergent or adishwasher detergent.

[0117] The invention will now be described in greater detail in thefollowing examples which are, however, not to be construed to limit theinvention.

EXAMPLES Example I Cloning of the Subtilisin E Gene and ExtracelluarExpression by B. subtilis

[0118] The aprE gene coding for subtilisin E was amplified by PCR fromthe genome of Bacillus subtilis strain 168 (DSM #402). Amplification wasperformed for 20 cycles using a 5:1 mixture of Taq and Pfu polymeraseswhile B. subtilis cells served as templates. The gene was ligated intothe vector pBV1 behind the P43 promotor. The vector pBV1 was constructedas follows: The pMB1 origin from pUC19 (ATCC 37254) was PCR amplified(positions 763-1601) and introduced into the PvuII site of pUB110 (ATCC37015). The fragment between SapI and BglII was removed from this vectorthereby deleting its mob gene (Colin R. Harwood, editor (1989) Bacillus,Plenum Press, New York). Then, an insert containing the P43 promoterfrom the cdd gene of B. subtilis, the signal sequence and the terminatorfrom the subtilisin E gene of B. subtilis, as well as a short multiplecloning site between the signal sequence and the terminator wasintroduced into the unique SphI site, resulting in the vector pBV1.

[0119] The wild type subtilisin E gene (Seq. ID:60) from the genome ofBacillus subtilis strain 168 (DSM #402) without the signal sequence aswell as any other subtilisin variant was introduced in frame with thesignal sequence into the multiple cloning site resulting in the vectorpBVP43-Sub (J. Biol. Chem. 1984, 259 (13), 8619-8625; Biotechnol.Bioeng. 1999, 62 (1), 87-96). Mutagenesis was restricted to the DNAsequence encoding the mature subtilisin (amino acid sequence ofsubtilisin E in Seq. ID: 2) without affecting the N-terminal signalsequence. The vector construct is schematically shown in FIG. 1.Identity of the intermediates as well as the final product of thecloning procedure was confirmed by means of DNA cycle sequencing usingthe didesoxynucleotide method. Functionality of the construct wasconfirmed by plating transformants of pBVP43-Sub in an aprE deficient B.subtilis strain (Harwood and Cutting (1990) Molecular Biological Methodsfor Bacillus, J. Wiley and Sons, New York) on LB agar containing 1% skimmilk and the appropriate antibiotics. Subtilisin activity resulted incleared halos around the Bacillus subtilis colonies. Expression ofsubtilisin E or a subtilisin variant was done by inoculating complexmedia containing NaCl, trypton and yeast extract with a aprE deficientB.

[0120] subtilis clone transformed with a pBVP43-Sub plasmid encoding theparticular variant, and incubation in Erlenmeyer flasks for 24 to 48 hat 30° C. with continuous shaking. After separation of the cells bycentrifugation, either the supernatant was used directly for enzymecharacterization, or the enzyme was further purified by saltprecipitation, ion exchange chromatography, dialysis and subsequentlyophilization. Concentration of the enzyme in solution was determinedby standard protein quantification methods such as the Bradford test.Purity and integrity of the final enzyme was analyzed by SDSpolyacrylamide gel electrophoresis.

Example II Generation of Improved Subtilisin Variants by Means ofDirected Evolution

[0121] Subtilisin variants with improved characteristics were generatedby means of directed evolution essentially following the protocolprovided in WO9218645 in combination with screening methods as providedin WO9416313 or WO9934195. According to these methods, the gene codingfor subtilisin E was mutagenized by replication using a DNA polymerasewith an error rate in the region of the error threshold. Typically, amodified PCR was used for this purpose. Ligation into the vector wasdone using 300 fmol vector, 1500 fmol insert, 2 μl of 10× Ligationbuffer (500 mM Tris-HCl, pH 7.5; 100 mM MgCl₂; 100 mM DTT; 10 mM ATP,250 μg/ml BSA), 5 Weiss Units of T4 DNA ligase (MBI Fermentas), ad 20 μlaqua dest, by incubation for 2 h at room temperature, followed by heatinactivation for 10 min at 65° C., and ethanol precipitation. Theligation mixture was then transformed into electrocompetent E. coliXL1-Blue. After preparation of the resulting DNA product from E. coliXL1-Blue cells, the library was transformed into an aprE deficient B.subtilis strain. Transformed B. subtilis cells containing the librarywere then distributed into the compartments of a suitable samplecarrier. Subtilisin variants produced by B. subtilis cells in theparticular compartments were then examined under conditions relevant tothe final application by ultra high throughput screening with confocalfluorescence spectroscopy (see WO9416313 or WO9934195). Genes coding forsubtilisin variants with improved characteristics under the screeningconditions were subsequently selected and isolated by means of PCRamplification, recloned and re-mutagenized. This cycle of mutagenesis,screening and selection was repeated several times. Optionally, thegenes coding for selected variants were analyzed by sequencing. As afurther option, mutations occurring in different variants selected in ascreening round were combined by means of standard restriction andligation methods or alternatively by means of homologous recombinationmethods, e.g. as provided in WO0134835. An example of the continuousimprovement of subtilisin characteristics by directed evolution over 4rounds is shown in FIG. 3.

Example III Protein Degradation Performance of Improved SubtilisinVariants in Detergent

[0122] The improvement of subtilisin variants was analyzed by measuringthe rate of hydrolysis of protein substrates. Bovine serum albumin (BSA,obtained from Sigma) as an exemplary globular protein was labeled withthe fluorescent dye TIRTC (obtained from Sigma) resulting in a labelingdegree of approximately one mol dye per mol protein. The degradation ofthe labelled protein was measured in solution by single-moleculeanisotropy, fluorescence correlation spectroscopy and fluorescenceintensity measurements using a confocal fluorescence spectroscopy set-up(see WO9416313). For further evaluation, FITC-labelled casein (caseinobtained from Sigma, FITC obtained from Fluka) was used as a substrate.For analyzing the characteristics of enzyme variants under washingconditions, tests were carried out in a freshly prepared typical laundrydetergent solution. In FIG. 4 the degradation of an improved subtilisinvariant is shown in comparison to the wild type as well as anothercommercially applied subtilisin, Subtilisin Carlsberg from B.licheniformisobtained from two sources, at typical concentrations. InFIG. 5 another set of four variants is compared in terms of the amountof enzyme needed to catalyze protein degradation with the same rate. Ascan be easily seen, the best variant out of this set has the samecatalytic activity at {fraction (1/100)} concentration compared to thewild type. Table I gives an overview of several variants and theirdegradation performance is measured as protein degradation in detergentafter a periode of 30 minutes normalized to the wild type. TABLE Isubtilisin/ relative protein SEQ ID NO degradation performance wildtype/2 1.00 variant 1/10 3.91 variant 2/11 1.90 variant 3/12 4.34variant 4/13 6.85 variant 5/14 2.18 variant 7/16 5.29 variant 8/17 2.26variant 9/18 2.06 variant 10/19 4.56 variant 11/20 4.26 variant 12/212.81 variant 13/22 1.84 variant 15/24 4.30 variant 16/25 6.57 variant17/26 5.85 variant 18/27 6.60 variant 19/28 6.35 variant 20/29 7.45variant 21/30 8.50 variant 22/31 8.37 variant 23/32 8.04 variant 24/337.63 variant 25/34 7.74 variant 26/35 7.79 variant 27/36 6.16 variant28/37 5.98 variant 29/38 5.75 variant 30/39 6.73 variant 31/40 5.95variant 32/41 6.77 variant 33/42 6.67 variant 34/43 7.24 variant 35/444.12 variant 36/45 6.89 variant 37/46 7.45 variant 38/47 6.13 variant39/48 7.22 variant 40/49 5.91 variant 41/50 5.73 variant 42/51 3.77variant 43/52 4.99 variant 44/53 3.36

[0123]FIG. 6 and Table II compare the Km of some of these variants withthe Km of the wild type enzyme. TABLE II Subtilisin/SEQ ID NO Km [μM]Subtilisin E (wild type)/2 194 Variant 1/10 117 Variant 4/13 71 Variant22/31 26

Example IV Effect of Improved Variants on Protein Solubilization andSubsequent Degradation from Textiles

[0124] In order to analyze the degradation of proteins bound to textilesurfaces and, thereby, the efficieny of a particular subtilisin variantin removing protein stains from soiled textiles, a solid phase assay wasused. Standard cotton (Nr. 10A, wfk Testgewebe GmbH, Christenfeld 10,D41379 Brüggen-Bracht, Germany) was impregnated with 20% hen eggsolution containing TIRTC labelled BSA. Then the cotton was cut intopieces, dried, washed three times with laundry detergent containing noprotease, dried again and then incubated with laundry detergentcontaining the particular subtilisin variant, or commercially availablesubtilisin variant, both at 2.5 mg/l, or no protease as control. Therelease of proteins from the textile and the further degradation insolution was then measured by single-molecule anisotropy, fluorescencecorrelation spectroscopy and fluorescence intensity measurements using aconfocal fluorescence spectroscopy set-up (see WO9416313). FIG. 7compares the rate of release of labelled protein fragments catalyzed bya particular variant with the rate of a comercially available protease.

1 60 1 275 PRT Bacillus amyloliquefaciens 1 Ala Gln Ser Val Pro Tyr GlyVal Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr ThrGly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser SerHis Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met Val Pro Ser GluThr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Ala GlyThr Val Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val AlaPro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly SerGly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile AlaAsn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser GlySer Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser GlyVal Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170175 Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met AlaSer 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys HisPro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Gln AsnThr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly LeuIle Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 2 275 PRT Bacillussubtilis 2 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro AlaLeu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala ValIle Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Arg GlyGly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly SerSer His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn SerIle Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala ValLys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Ile Ile AsnGly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn MetSer Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val ValAsp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala Ala GlyAsn Glu Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly Tyr ProAla Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn Ser SerAsn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu Asp ValMet Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Gly ThrTyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His Val AlaGly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 Trp ThrAsn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 LeuGly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270Ala Ala Gln 275 3 275 PRT Bacillus subtilis 3 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGlu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 4 274 PRT Bacilluslicheniformis 4 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala AspLys Val 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val AlaVal Leu Asp 20 25 30 Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val ValGly Gly Ala 35 40 45 Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly AsnGly His Gly 50 55 60 Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn ThrThr Gly Val 65 70 75 80 Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala ValLys Val Leu Asn 85 90 95 Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val SerGly Ile Glu Trp 100 105 110 Ala Thr Thr Asn Gly Met Asp Val Ile Asn MetSer Leu Gly Gly Ala 115 120 125 Ser Gly Ser Thr Ala Met Lys Gln Ala ValAsp Asn Ala Tyr Ala Arg 130 135 140 Gly Val Val Val Val Ala Ala Ala GlyAsn Ser Gly Asn Ser Gly Ser 145 150 155 160 Thr Asn Thr Ile Gly Tyr ProAla Lys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp Ser AsnSer Asn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu Leu Glu ValMet Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr 195 200 205 Pro Thr Asn ThrTyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 His Val AlaGly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu 225 230 235 240 SerAla Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu 245 250 255Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala 260 265270 Ala Gln 5 269 PRT Bacillus lentus 5 Ala Gln Ser Val Pro Trp Gly IleSer Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr GlySer Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His ProAsp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro SerThr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile AlaAla Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser AlaGlu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser Gly Ser ValSer Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly MetHis Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala ThrLeu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu ValVal Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser 145 150 155 160 TyrPro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val GlnIle 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly SerThr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala ThrArg 260 265 6 275 PRT Bacillus stearothermophilus 6 Ala Gln Ser Val ProTyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln GlyTyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile AspSer Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val ProSer Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His ValAla Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu GlyVal Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser ThrGly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp AlaIle Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 ProSer Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150155 160 Ser Ser Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser SerAla 180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile GlnSer Thr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr SerMet Ala Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu SerLys His Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg LeuGlu Ser Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly LysGly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 7 268 PRTBacillus sp. YaB 7 Gln Thr Val Pro Trp Gly Ile Asn Arg Val Gln Ala ProIle Ala Gln 1 5 10 15 Ser Arg Gly Phe Thr Gly Thr Gly Val Arg Val AlaVal Leu Asp Thr 20 25 30 Gly Ile Ser Asn His Ala Asp Leu Arg Ile Arg GlyGly Ala Ser Phe 35 40 45 Val Pro Gly Glu Pro Asn Ile Ser Asp Gly Asn GlyHis Gly Thr Gln 50 55 60 Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser IleGly Val Leu Gly 65 70 75 80 Val Ala Pro Asn Val Asp Leu Tyr Gly Val LysVal Leu Gly Ala Ser 85 90 95 Gly Ser Gly Ser Ile Ser Gly Ile Ala Gln GlyLeu Gln Trp Ala Ala 100 105 110 Asn Asn Gly Met His Ile Ala Asn Met SerLeu Gly Ser Ser Ala Gly 115 120 125 Ser Ala Thr Met Glu Gln Ala Val AsnGln Ala Thr Ala Ser Gly Val 130 135 140 Leu Val Val Ala Ala Ser Gly AsnSer Gly Ala Gly Asn Val Gly Phe 145 150 155 160 Pro Ala Arg Tyr Ala AsnAla Met Ala Val Gly Ala Thr Asp Gln Asn 165 170 175 Asn Asn Arg Ala ThrPhe Ser Gln Tyr Gly Ala Gly Leu Asp Ile Val 180 185 190 Ala Pro Gly ValGly Val Gln Ser Thr Val Pro Gly Asn Gly Tyr Ala 195 200 205 Ser Phe AsnGly Thr Ser Met Ala Thr Pro His Val Ala Gly Val Ala 210 215 220 Ala LeuVal Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg 225 230 235 240Asn His Leu Lys Asn Thr Ala Thr Asn Leu Gly Asn Thr Thr Gln Phe 245 250255 Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 8 380 PRTBacillus alcalophilus 8 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser ThrAla Leu Leu Ile 1 5 10 15 Ser Val Ala Phe Ser Ser Ser Ile Ala Ser AlaAla Glu Glu Ala Lys 20 25 30 Glu Lys Tyr Leu Ile Gly Phe Asn Glu Gln GluAla Val Ser Glu Phe 35 40 45 Val Glu Gln Val Glu Ala Asn Asp Glu Val AlaIle Leu Ser Glu Glu 50 55 60 Glu Glu Val Glu Ile Glu Leu Leu His Glu PheGlu Thr Ile Pro Val 65 70 75 80 Leu Ser Val Glu Leu Ser Pro Glu Asp ValAsp Ala Leu Glu Leu Asp 85 90 95 Pro Ala Ile Ser Tyr Ile Glu Glu Asp AlaGlu Val Thr Thr Met Ala 100 105 110 Gln Ser Val Pro Trp Gly Ile Ser ArgVal Gln Ala Pro Ala Ala His 115 120 125 Asn Arg Gly Leu Thr Gly Ser GlyVal Lys Val Ala Val Leu Asp Thr 130 135 140 Gly Ile Ser Thr His Pro AspLeu Asn Ile Arg Gly Gly Ala Ser Phe 145 150 155 160 Val Pro Gly Glu ProSer Thr Gln Asp Gly Asn Gly His Gly Thr His 165 170 175 Val Ala Gly ThrIle Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly 180 185 190 Val Ala ProAsn Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala Ser 195 200 205 Gly SerGly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly 210 215 220 AsnAsn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser Pro 225 230 235240 Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly Val 245250 255 Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser Tyr260 265 270 Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp GlnAsn 275 280 285 Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu AspIle Val 290 295 300 Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly SerThr Tyr Ala 305 310 315 320 Ser Leu Asn Gly Thr Ser Met Ala Thr Pro HisVal Ala Gly Ala Ala 325 330 335 Ala Leu Val Lys Gln Lys Asn Pro Ser TrpSer Asn Val Gln Ile Arg 340 345 350 Asn His Leu Lys Asn Thr Ala Thr SerLeu Gly Ser Thr Asn Leu Tyr 355 360 365 Gly Ser Gly Leu Val Asn Ala GluAla Ala Thr Arg 370 375 380 9 380 PRT Bacillus clausii 9 Met Lys Lys ProLeu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile 1 5 10 15 Ser Val AlaPhe Ser Ser Ser Ile Ala Ser Ala Ala Glu Glu Ala Lys 20 25 30 Glu Lys TyrLeu Ile Gly Phe Asn Glu Gln Glu Ala Val Ser Glu Phe 35 40 45 Val Glu GlnVal Glu Ala Asn Asp Glu Val Ala Ile Leu Ser Glu Glu 50 55 60 Glu Glu ValGlu Ile Glu Leu Leu His Glu Phe Glu Thr Ile Pro Val 65 70 75 80 Leu SerVal Glu Leu Ser Pro Glu Asp Val Asp Ala Leu Glu Leu Asp 85 90 95 Pro AlaIle Ser Tyr Ile Glu Glu Asp Ala Glu Val Thr Thr Met Ala 100 105 110 GlnSer Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His 115 120 125Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp Thr 130 135140 Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser Phe 145150 155 160 Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly ThrHis 165 170 175 Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly ValLeu Gly 180 185 190 Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val LeuGly Ala Ser 195 200 205 Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly LeuGlu Trp Ala Gly 210 215 220 Asn Asn Gly Met His Val Ala Asn Leu Ser LeuGly Ser Pro Ser Pro 225 230 235 240 Ser Ala Thr Leu Glu Gln Ala Val AsnSer Ala Thr Ser Arg Gly Val 245 250 255 Leu Val Val Ala Ala Ser Gly AsnSer Gly Ala Gly Ser Ile Ser Tyr 260 265 270 Pro Ala Arg Tyr Ala Asn AlaMet Ala Val Gly Ala Thr Asp Gln Asn 275 280 285 Asn Asn Arg Ala Ser PheSer Gln Tyr Gly Ala Gly Leu Asp Ile Val 290 295 300 Ala Pro Gly Val AsnVal Gln Ser Thr Tyr Pro Gly Ser Thr Tyr Ala 305 310 315 320 Ser Leu AsnGly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala Ala 325 330 335 Ala LeuVal Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg 340 345 350 AsnHis Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr 355 360 365Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 370 375 380 10 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 1 10 Ala Gln Ser Val Pro Asn Gly Ile Ser Gln Ile Lys Ala Pro AlaLeu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala ValLeu Asp 20 25 30 Ser Gly Ile Asp Ser Thr His Pro Asp Leu Asn Val Arg GlyGly Ala 35 40 45 Ser Ile Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly SerSer His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn SerIle Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala ValLys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile Ile AsnGly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn MetSer Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val ValAsp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala Ala GlyAsn Glu Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly Tyr ProAla Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn Ser SerAsn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu Asp ValMet Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Gly ThrTyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His Val AlaGly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 Trp ThrAsn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 LeuGly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270Ala Ala Gln 275 11 275 PRT Artificial Sequence Description of ArtificialSequence Subtilisin E variant 2 11 Ala Gln Ser Val Pro Tyr Gly Ile SerGln Ile Lys Ala Thr Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly SerAsn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His ProAsp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr AsnPro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val Ala Gly Thr IleAla Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro SerAla Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly ArgTyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn AsnMet Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ile Gly Ser ThrAla Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile ValVal Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150 155 160 Ser ThrSer Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 ValGly Ala Val Asn Ser Ser Asn His Arg Ala Ser Phe Ser Ser Ala 180 185 190Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr AlaThr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Asn Gly Leu Ile AsnVal Gln Ala 260 265 270 Ala Ala Gln 275 12 275 PRT Artificial SequenceDescription of Artificial Sequence Subtilisin E variant 3 12 Ala Gln SerVal Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His SerGln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser GlyIle Asp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser PheIle Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly ThrHis Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 ValLeu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 AspSer Thr Gly Ser Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130135 140 Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly145 150 155 160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser ThrIle Ala 165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser PheSer Ser Ala 180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val SerIle Gln Ser Thr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn GlyThr Ser Met Ala Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu IleLeu Ser Lys His Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg AspArg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr TyrGly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 13 275PRT Artificial Sequence Description of Artificial Sequence Subtilisin Evariant 4 13 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro AlaLeu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala ValIle Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Arg GlyGly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly SerSer His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn SerIle Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala ValLys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile Ile AsnGly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn MetSer Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Ala ValAsp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Val Ala Ala Ala GlyAsn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly Tyr ProAla Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn Ser SerAsn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu Asp ValMet Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Gly ThrTyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His Val AlaGly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 Trp ThrAsn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 LeuGly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val His Ala 260 265 270Ala Ala Gln 275 14 275 PRT Artificial Sequence Description of ArtificialSequence Subtilisin E variant 5 14 Ala Gln Ser Val Pro Tyr Gly Ile ProGln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly SerAsn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His ProAsp Leu Asn Val Ser Gly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr AsnPro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val Ala Gly Thr IleAla Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro SerAla Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly ArgTyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn AsnMet Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ala Gly Ser ThrAla Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile ValVal Ala Ala Ala Ala Gly Asn Gly Gly Ser Ser Gly 145 150 155 160 Ser ThrSer Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 ValGly Ala Val Asn Ser Asn Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr AlaThr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile AsnVal Gln Ala 260 265 270 Ala Ala Gln 275 15 275 PRT Artificial SequenceDescription of Artificial Sequence Subtilisin E variant 6 15 Ala Gln SerVal Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His SerGln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser GlyIle Asp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser PheVal Pro Ser Glu Thr Asn Pro Tyr Gln Asp Ser Ser Ser His 50 55 60 Gly ThrHis Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Pro Ile Gly 65 70 75 80 ValLeu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 AspSer Thr Gly Ser Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130135 140 Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly145 150 155 160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser ThrIle Ala 165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser PheSer Ser Ala 180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val SerIle Gln Ser Thr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn GlyThr Ser Met Ala Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu IleLeu Ser Lys His Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg AspArg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr TyrGly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 16 275PRT Artificial Sequence Description of Artificial Sequence Subtilisin Evariant 7 16 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro AlaLeu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala ValIle Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val Arg GlyGly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp Gly SerSer His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn SerPhe Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala ValLys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile Ile AsnGly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn MetSer Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr Val ValAsp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala Ala GlyAsn Gly Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly Tyr ProAla Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn Ser SerAsn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu Asp ValMet Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Gly ThrTyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His Val AlaGly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 Trp ThrSer Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 LeuGly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270Ala Ala Gln 275 17 275 PRT Artificial Sequence Description of ArtificialSequence Subtilisin E variant 8 17 Ala Gln Ser Val Pro Tyr Gly Ile SerGln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly SerAsn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His ProAsp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr AsnPro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val Ala Gly Thr IleAla Ala Leu Asn Asn Pro Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro SerAla Ser Ile Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly ArgTyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn AsnMet Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser ThrAla Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile ValVal Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser ThrSer Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 ValGly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr AlaThr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile AsnVal Gln Ala 260 265 270 Ala Ala Gln 275 18 275 PRT Artificial SequenceDescription of Artificial Sequence Subtilisin E variant 9 18 Ala Gln SerVal Pro Tyr Gly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His SerGln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser GlyIle Asp Ser Ser His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser PheVal Pro Ser Glu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly ThrHis Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 ValLeu Gly Val Ser Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 AspSer Thr Gly Ser Gly Lys Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110Trp Ala Ile Ser Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120125 Pro Ala Gly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130135 140 Ser Gly Ile Val Val Ala Ala Ala Ala Gly Asn Gly Gly Ser Ser Gly145 150 155 160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser ThrIle Ala 165 170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser PheSer Ser Ala 180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val SerIle Gln Ser Thr 195 200 205 His Pro Gly Gly Thr Tyr Gly Ala Tyr Asn GlyThr Ser Met Ala Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu IleLeu Ser Lys His Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg AspArg Leu Glu Ser Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr TyrGly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 19 275PRT Artificial Sequence Description of Artificial Sequence Subtilisin Evariant 10 19 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Lys Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpAla Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly His Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 20 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 11 20 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asp Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGlu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Lys Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 21 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 12 21 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ThrGly Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Lys Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Ala Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Gly Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Gly Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Leu Ala 260 265270 Ala Ala Gln 275 22 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 13 22 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGlu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Lys Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Lys Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Ser Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluGly Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Leu Ala 260 265 270 Ala Ala Gln 275 23 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 14 23 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Ile Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp SerSer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnPro Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Lys Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Ala Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Gly Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Thr Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 24 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 15 24 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Ser Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 GlyGly Ile Val Val Ala Ala Ala Ala Gly Asn Lys Asp Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Val Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Val Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 25 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 16 25 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Thr Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Ser Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr AlaVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Val Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Leu Ala 260 265270 Ala Ala Gln 275 26 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 17 26 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Thr Val Leu 1 5 10 15 Leu Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGlu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro AlaGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 27 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 18 27 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr AlaVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Val Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val His Ala 260 265270 Ala Ala Gln 275 28 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 19 28 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGlu Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Ser Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Thr Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Ala 210 215 220 Pro His Val Thr Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 29 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 20 29 Ala Gln Ser Ala Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp SerSer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerAsn Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 30 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 21 30 Ala Gln Ser Val Pro TyrGly Ile Pro Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGly Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Val Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 31 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 22 31 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Lys Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 32 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 23 32 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Thr Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGlu Thr Asn Pro Tyr Gln Asp Ser Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Asn Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Ser Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 33 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 24 33 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Asp Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Thr Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Ala Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerAsn Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Ile Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 34 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 25 34 Ala Gln Ser Ala Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Ser Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 GlyGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Asn Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 35 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 26 35 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr AlaVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Val Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Val Ser Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 36 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 27 36 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Thr Val Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Leu Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Ser Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 37 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 28 37 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser His Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Leu Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Leu Asp GlySer Pro His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Asn Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Ala Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Gly Gly Ser Ser Gly 145 150 155 160 Asn Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerAsn Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr His Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Phe Ile His Val Gln Ala 260 265270 Ala Ala Gln 275 38 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 29 38 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Asn Glu 100 105 110 Trp Ala IleSer Asn Tyr Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro AlaGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn His Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Asn Pro Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Leu Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly His Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 39 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 30 39 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Asn Glu 100 105 110 Trp Ala Ile Ser Asn Tyr Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Ala Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn His Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 40 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 31 40 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro AlaGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Ile Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Asn Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Gly Val Met Ala Pro Gly Val Ser Ile His SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Ile Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Ile Val Gln Ala 260 265 270 Ala Ala Gln 275 41 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 32 41 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Lys Ala Leu Asn Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asp Val Gln Ala 260 265270 Ala Ala Gln 275 42 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 33 42 Ala Gln Ser Val Pro AsnGly Ile Ser Gln Ile Lys Ala Thr Val Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Leu Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Ser Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 43 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 34 43 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ThrVal Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly PhePro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Thr Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 44 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 35 44 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Thr Val Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Leu Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Tyr Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Gly Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Thr Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Cys Gly Lys GlyLeu Ile His Val Gln Ala 260 265 270 Ala Ala Gln 275 45 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 36 45 Ala Gln Ser Val Pro Tyr Ser Ile Ala Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Lys Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Ala 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Asn Gly Lys Gly Leu Ile Asn Val Gln Pro 260 265270 Ala Ala Gln 275 46 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 37 46 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Lys Ala Leu Asn Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 47 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 38 47 Ala Gln Ser Val Pro Tyr Gly Ile Pro Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Lys Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Ser Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerAsn Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 48 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 39 48 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Lys Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Tyr Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Leu SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Asn GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 49 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 40 49 Ala Gln Ser Val Leu His Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Gly Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Lys Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 50 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 41 50 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGly Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Val Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Ala Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Val Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Ile Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 51 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 42 51 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Pro Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val His Ala 260 265270 Ala Ala Gln 275 52 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 43 52 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Leu Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Ser Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerGlu Thr Ser Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Ala Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Val Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val His Ala 260 265 270 Ala Ala Gln 275 53 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 44 53 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ThrVal Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Ala Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala His Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val His Ala 260 265270 Ala Ala Gln 275 54 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 45 54 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Lys Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Ala Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 55 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 46 55 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Lys Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Glu Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 56 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 47 56 Ala Gln Ser Ala Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Asn Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Ser Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 SerGly Ile Val Val Ala Ala Ala Ala Gly Asn Glu Gly Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Asn Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 57 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 48 57 Ala Gln Ser Val Pro Tyr Gly Ile Pro Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Asp Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Arg Tyr Ser Trp Ile IleAsn Gly Val Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala Ala Ala AlaGly Asn Glu Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerAsn Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val Arg Val Ser Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 58 275 PRT Artificial Sequence Description ofArtificial Sequence Subtilisin E variant 49 58 Ala Gln Ser Val Pro TyrGly Ile Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly TyrThr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp SerSer His Pro Asp Leu Ser Val Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro SerAsp Thr Asn Pro Tyr Gln Asp Gly Ser Ser His 50 55 60 Gly Thr His Val AlaGly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly ValSer Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr GlySer Gly Arg Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala IleSer Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro ThrGly Ser Thr Ala Leu Lys Thr Val Val Asp Lys Ala Val Ser 130 135 140 GlyGly Ile Val Val Ala Ala Ala Ala Gly Asn Lys Asp Ser Ser Gly 145 150 155160 Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165170 175 Val Gly Ala Val Asn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala180 185 190 Gly Ser Glu Leu Asp Val Val Ala Pro Gly Val Ser Ile Gln SerThr 195 200 205 Leu Pro Gly Gly Thr Tyr Gly Ala His Asn Gly Thr Ser MetAla Thr 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser LysHis Pro Thr 225 230 235 240 Trp Thr Asn Ala Gln Val Arg Val Arg Leu GluSer Thr Ala Thr Tyr 245 250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys GlyLeu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 59 275 PRTArtificial Sequence Description of Artificial Sequence Subtilisin Evariant 50 59 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile Lys Ala ProAla Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val AlaVal Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Asn Val ArgGly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gln Asp GlySer Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala Leu Asn AsnSer Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser Leu Tyr AlaVal Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Ile IleAsn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp Val Ile AsnMet Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu Lys Thr ValVal Asp Lys Ala Val Ser 130 135 140 Gly Gly Ile Val Val Ala Ala Ala AlaGly Asn Lys Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr Val Gly TyrPro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala Val Asn SerSer Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser Glu Leu AspVal Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly GlyThr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 Pro His ValAla Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235 240 TrpThr Asn Ala Gln Val His Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245 250 255Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265270 Ala Ala Gln 275 60 1146 DNA Bacillus subtilis 60 gtgagaagcaaaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60 gcgttcagcaacatgtctgt gcaggctgcc ggaaaaagca gtacagaaaa gaaatacatt 120 gtcggatttaaacagacaat gagtgccatg agttccgcca agaaaaagga tgttatttct 180 gaaaaaggcggaaaggttca aaagcaattt aagtatgtta acgcggccgc agcaacattg 240 gatgaaaaagctgtaaaaga attgaaaaaa gatccgagcg ttgcatatgt ggaagaagat 300 catattgcacatgaatatgc gcaatctgtt ccttatggca tttctcaaat taaagcgccg 360 gctcttcactctcaaggcta cacaggctct aacgtaaaag tagctgttat cgacagcgga 420 attgactcttctcatcctga cttaaacgtc agaggcggag caagcttcgt accttctgaa 480 acaaacccataccaggacgg cagttctcac ggtacgcatg tagccggtac gattgccgct 540 cttaataactcaatcggtgt tctgggcgtt agcccaagcg catcattata tgcagtaaaa 600 gtgcttgattcaacaggaag cggccaatat agctggatta ttaacggcat tgagtgggcc 660 atttccaacaatatggatgt tatcaacatg agccttggcg gacctactgg ttctacagcg 720 ctgaaaacagtcgttgacaa agccgtttcc agcggtatcg tcgttgctgc cgcagccgga 780 aacgaaggttcatccggaag cacaagcaca gtcggctacc ctgcaaaata tccttctact 840 attgcagtaggtgcggtaaa cagcagcaac caaagagctt cattctccag cgcaggttct 900 gagcttgatgtgatggctcc tggcgtgtcc atccaaagca cacttcctgg aggcacttac 960 ggcgcttataacggaacgtc catggcgact cctcacgttg ccggagcagc agcgttaatt 1020 ctttctaagcacccgacttg gacaaacgcg caagtccgtg atcgtttaga aagcactgca 1080 acatatcttggaaactcttt ctactatgga aaagggttaa tcaacgtaca agcagctgca 1140 caataa 1146

1. A modified subtilisin which has at least one mutation in an aminoacid sequence of a precursor subtilisin at a position numbered accordingto the amino acid sequence of the mature subtilisin BPN′ shown in SEQ IDNO:1, wherein said at least one mutation is selected from (a) E54D orE54G, (b) Q103R or Q103K, (c) T133K, (d) E156K or E156A, and (e) Y217H,or mutations at the respective positions which are homologous to saidmutations (a) to (e), provided that in case of mutations E54D, Q103R,E156K or E156A the modified subtilisin has at least one more of saidmutations (a) to (e).
 2. The modified subtilisin of claim 1, which hasat least two (2) of said mutations (a) to (e).
 3. The modified subtilisnof claim 1, which has at least three (3) of said mutations (a) to (e).4. The modified subtilisins of claim 1, which has at least four (4) ofsaid mutations (a) to (e).
 5. The modified subtilisins of claim 1, whichhas at least five (5) of said mutations (a) to (e).
 6. The modifiedsubtilisin of claim 1, which has at least two (2) of the following groupof amino acid substitutions: E54D, Q103R, T133K, E156A, Y217H.
 7. Themodified subitilisn of claim 1, which has at least three (3) of thefollowing group of amino acid substitutions: E54D, Q103R, T133K, E156A,Y217H.
 8. The modified subtilisins of claim 1, which has at least four(4) of the following group of amino acid substitutions: E54D, Q103R,T133K, E156A, Y217H.
 9. The modified subtilisins of claim 1, which hasat least five (5) of the following group of amino acid substitutions:E54D, Q103R, T133K, E156A, Y217H.
 10. The modified subtilisin of any oneof claim 1, which further has mutation at the following amino acidresidue positions of the precursor subtilisin: 4, 5, 6, 7, 9, 14, 15,17, 18, 19, 20, 24, 25, 31, 38, 43, 45, 49, 50, 51, 54 modifieddifferently as compared to mutation (a), 56, 59, 61, 63, 78, 79, 89, 90,101, 103 modified differently as compared to mutation (b), 104, 111,118, 130, 133 modified differently as compared to mutation (c), 136,138, 145, 149, 150, 156 modified differently as compared to mutation(d), 157, 161, 164, 167, 173, 181, 183, 184, 185, 191, 197, 199, 204,206, 209, 217 modified differently as compared to mutation (e), 218,224, 228, 234, 235, 242, 243, 245, 247, 248, 249, 251, 252, 253, 259,263, 265, 271, 267, 269, 271, 272, 273 and
 275. 11. The modifiedsubtilisin of claim 1, which further has one or more of the followingmutations: V4A, P5L, Y6N, Y6H, G7S, S9P, S9A, P14T, A15G, A15V, H17L,S18T, Q19H, G20D, S24T, N25D, I31L, S38T, N43S, N43I, N43T, R45S, S49G,F50I, V51I, N56S, Q59L, G61S, S63P, S78P, I79F, S89P, L90I, Y104N,I111V, I111N, N118Y, T130I, T130A, K136N, V138A, S145G, V149I, A150V,E156G, G157D, S161N, T164S, Y167F, N181S, S183N, N184Y, Q185H, S191G,D197G, M199V, S204T, Q206H, Q206L, L209H, N218I, N218T, T224A, A228T,I234N, L235I, L235P, T242A, N243S, N243H, N243T, Q245L, R247H, D248V,R249S, S252G, T253S, N259H, Y263C, Y263N, K265N, L267F, N269H, N269I,N269D, Q271H, Q271L, A272P and Q275L.
 12. The modified subtilisinaccording to claim 1, which has a mutation (a) and has at least one (1)additional mutations selected from the mutations (b) to (e).
 13. Themodified subtilisins according to claim 12, which has at least two (2)additional mutations selected from the mutations (b) to (e).
 14. Themodified subtilisins according to claim 12, which has at least three (3)additional mutations selected from the mutations (b) to (e).
 15. Themodified subtilisins according to claim 12, which has at least four (4)additional mutations selected from the mutations (b) to (e).
 16. Themodified subtilisin according to claim 12, which has at least theadditional mutations Q103R and E156A.
 17. The modified subitlisin ofclaim 12, which has the modification E54D.
 18. The modified subtilisinaccording to claim 14, which has at least the additional mutations Q103Rand E156A.
 19. The modified subtilisin of any one of claim 12, whichfurther has mutation at the following amino acid residue positions ofthe precursor subtilisin: 4, 5, 6, 7, 9, 14, 15, 17, 18, 19, 20, 24, 25,31, 38, 43, 45, 49, 50, 51, 54 modified differently as compared tomutation (a), 56, 59, 61, 63, 78, 79, 89, 90, 101, 103 modifieddifferently as compared to mutation (b), 104, 111, 118, 130, 133modified differently as compared to mutation (c), 136, 138, 145, 149,150, 156 modified differently as compared to mutation (d), 157, 161,164, 167, 173, 181, 183, 184, 185, 191, 197, 199, 204, 206, 209, 217modified differently as compared to mutation (e), 218, 224, 228, 234,235, 242, 243, 245, 247, 248, 249, 251, 252, 253, 259, 263, 265, 271,267, 269, 271, 272, 273 and
 275. 20. The modified subtilisin of claim12, which further has one or more of the following mutations: V4A, P5L,Y6N, Y6H, G7S, S9P, S9A, P14T, A15G, A15V, H17L, S18T, Q19H, G20D, S24T,N25D, I31L, S38T, N43S, N43I, N43T, R45S, S49G, F50I, V51I, N56S, Q59L,G61S, S63P, S78P, I79F, S89P, L90I, Y104N, I111V, I111N, N118Y, T130I,T130A, K136N, V138A, S145G, V149I, A150V, E156G, G157D, S161N, T164S,Y167F, N181S, S183N, N184Y, Q185H, S191G, D197G, M199V, S204T, Q206H,Q206L, L209H, N218I, N218T, T224A, A228T, I234N, L235I, L235P, T242A,N243S, N243H, N243T, Q245L, R247H, D248V, R249S, S252G, T253S, N259H,Y263C, Y263N, K265N, L267F, N269H, N269I, N269D, Q271H, Q271L, A272P andQ275L.
 21. The modified subtilisin according to claim 1, which has amutation (b) and has at least one (1) additional mutation selected fromthe mutations (a) and (c) to (e).
 22. The modified subtilisins accordingto claim 21, which has at least two (2) additional mutations selectedfrom the mutations (a) and (c) to (e).
 23. The modified subtilisinsaccording to claim 21, which has at least three (3) additional mutationsselected from the mutations (a) and (c) to (e).
 24. The modifiedsubtilisins according to claim 21, which has at least four (4)additional mutations selected from the mutations (a) and (c) to (e). 25.The modified subtilisins according to claim 21, which has themodification Q103R.
 26. The modified subtilisin according to claim 21,which has modifications Q103R and E156A and at least one furthermodification (a), (c) or (e).
 27. The modified subtilisins according toclaim 26, which has the further mutation T133K.
 28. The modifiedsubtilisin of any one of claim 21, which further has mutation at thefollowing amino acid residue positions of the precursor subtilisin: 4,5, 6, 7, 9, 14, 15, 17, 18, 19, 20, 24, 25, 31, 38, 43, 45, 49, 50, 51,54 modified differently as compared to mutation (a), 56, 59, 61, 63, 78,79, 89, 90, 101, 103 modified differently as compared to mutation (b),104, 111, 118, 130, 133 modified differently as compared to mutation(c), 136, 138, 145, 149, 150, 156 modified differently as compared tomutation (d), 157, 161, 164, 167, 173, 181, 183, 184, 185, 191, 197,199, 204, 206, 209, 217 modified differently as compared to mutation(e), 218, 224, 228, 234, 235, 242, 243, 245, 247, 248, 249, 251, 252,253, 259, 263, 265, 271, 267, 269, 271, 272, 273 and
 275. 29. Themodified subtilisin of claim 21, which further has one or more of thefollowing mutations: V4A, P5L, Y6N, Y6H, G7S, S9P, S9A, P14T, A15G,A15V, H17L, S18T, Q19H, G20D, S24T, N25D, I31L, S38T, N43S, N43I, N43T,R45S, S49G, F50I, V51I, N56S, Q59L, G61S, S63P, S78P, I79F, S89P, L90I,Y104N, I111V, I111N, N118Y, T130I, T130A, K136N, V138A, S145G, V149I,A150V, E156G, G157D, S161N, T164S, Y167F, N181S, S183N, N184Y, Q185H,S191G, D197G, M199V, S204T, Q206H, Q206L, L209H, N218I, N218T, T224A,A228T, 1234N, L235I, L235P, T242A, N243S, N243H, N243T, Q245L, R247H,D248V, R249S, S252G, T253S, N259H, Y263C, Y263N, K265N, L267F, N269H,N269I, N269D, Q271H, Q271L, A272P and Q275L.
 30. The modified subtilisinaccording to claim 1, which has modification (c) and has at least one(1) additional mutation selected from the mutations (a), (b), (d) and(e).
 31. The modified subtilisin according to claim 1, which hasmodification (c) and has at least two (2) additional mutations selectedfrom the mutations (a), (b), (d) and (e).
 32. The modified subtilisinaccording to claim 1, which has modification (c) and has at least three(3) additional mutations selected from the mutations (a), (b), (d) and(e).
 33. The modified subtilisin according to claim 1, which hasmodification (c) and has at least four (4) additional mutations selectedfrom the mutations (a), (b), (d) and (e).
 34. The modified subtilisin ofany one of claim 30, which further has mutation at the following aminoacid residue positions of the precursor subtilisin: 4, 5, 6, 7, 9, 14,15, 17, 18, 19, 20, 24, 25, 31, 38, 43, 45, 49, 50, 51, 54 modifieddifferently as compared to mutation (a), 56, 59, 61, 63, 78, 79, 89, 90,101, 103 modified differently as compared to mutation (b), 104, 111,118, 130, 133 modified differently as compared to mutation (c), 136,138, 145, 149, 150, 156 modified differently as compared to mutation(d), 157, 161, 164, 167, 173, 181, 183, 184, 185, 191, 197, 199, 204,206, 209, 217 modified differently as compared to mutation (e), 218,224, 228, 234, 235, 242, 243, 245, 247, 248, 249, 251, 252, 253, 259,263, 265, 271, 267, 269, 271, 272, 273 and
 275. 35. The modifiedsubtilisin of claim 30, which further has one or more of the followingmutations: V4A, P5L, Y6N, Y6H, G7S, S9P, S9A, P14T, A15G, A15V, H17L,S18T, Q19H, G20D, S24T, N25D, I31L, S38T, N43S, N43I, N43T, R45S, S49G,F50I, V51I, N56S, Q59L, G61S, S63P, S78P, I79F, S89P, L90I, Y104N,I111V, I111N, N118Y, T130I, T130A, K136N, V138A, S145G, V149I, A150V,E156G, G157D, S161N, T164S, Y167F, N181S, S183N, N184Y, Q185H, S191G,D197G, M199V, S204T, Q206H, Q206L, L209H, N218I, N218T, T224A, A228T,I234N, L235I, L235P, T242A, N243S, N243H, N243T, Q245L, R247H, D248V,R249S, S252G, T253S, N259H, Y263C, Y263N, K265N, L267F, N269H, N269I,N269D, Q271H, Q271L, A272P and Q275L.
 36. The modified subtilisinaccording to claim 1, which has a mutation (d) and has at least one (1)additional mutation selected from the mutations (a) to (c) and (e). 37.The modified subtilisin according to claim 36, which has a mutation (d)and has at least two (2) additional mutations selected from themutations (a) to (c) and (e).
 38. The modified subtilisin according toclaim 36, which has a mutation (d) and has at least three (3) additionalmutations selected from the mutations (a) to (c) and (e).
 39. Themodified subtilisin according to claim 36, which has a mutation (d) andhas at least four (4) additional mutations selected from the mutations(a) to (c) and (e).
 40. The modified subtilisins according to claim 36,which has the modification E156A.
 41. The modified subtilisin of any oneof claim 36, which further has mutation at the following amino acidresidue positions of the precursor subtilisin: 4, 5, 6, 7, 9, 14, 15,17, 18, 19, 20, 24, 25, 31, 38, 43, 45, 49, 50, 51, 54 modifieddifferently as compared to mutation (a), 56, 59, 61, 63, 78, 79, 89, 90,101, 103 modified differently as compared to mutation (b), 104, 111,118, 130, 133 modified differently as compared to mutation (c), 136,138, 145, 149, 150, 156 modified differently as compared to mutation(d), 157, 161, 164, 167, 173, 181, 183, 184, 185, 191, 197, 199, 204,206, 209, 217 modified differently as compared to mutation (e), 218,224, 228, 234, 235, 242, 243, 245, 247, 248, 249, 251, 252, 253, 259,263, 265, 271, 267, 269, 271, 272, 273 and
 275. 42. The modifiedsubtilisin of claim 37, which further has one or more of the followingmutations: V4A, P5L, Y6N, Y6H, G7S, S9P, S9A, P14T, A15G, A15V, H17L,S18T, Q19H, G20D, S24T, N25D, I31L, S38T, N43S, N43I, N43T, R45S, S49G,F50I, V51I, N56S, Q59L, G61S, S63P, S78P, I79F, S89P, L90I, Y104N,I111V, I111N, N118Y, T130I, T130A, K136N, V138A, S145G, V149I, A150V,E156G, G157D, S161N, T164S, Y167F, N181S, S183N, N184Y, Q185H, S191G,D197G, M199V, S204T, Q206H, Q206L, L209H, N218I, N218T, T224A, A228T,1234N, L235I, L235P, T242A, N243S, N243H, N243T, Q245L, R247H, D248V,R249S, S252G, T253S, N259H, Y263C, Y263N, K265N, L267F, N269H, N269I,N269D, Q271H, Q271L, A272P and Q275L.
 43. The modified subtilisinaccording to claim 1, which has a mutation (e) and has at least one (1)additional mutation selected from the mutations (a) to (d).
 44. Themodified subtilisin according to claim 43, which has a mutation (e) andhas at least two (2) additional mutations selected from the mutations(a) to (d).
 45. The modified subtilisin according to claim 43, which hasa mutation (e) and has at least three (3) additional mutations selectedfrom the mutations (a) to (d).
 46. The modified subtilisin according toclaim 43, which has a mutation (e) and has at least four (4) additionalmutations selected from the mutations (a) to (d).
 47. The modifiedsubtilisin of claim 43, which further has mutation at the followingamino acid residue positions of the precursor subtilisin: 4, 5, 6, 7, 9,14, 15, 17, 18, 19, 20, 24, 25, 31, 38, 43, 45, 49, 50, 51, 54 modifieddifferently as compared to mutation (a), 56, 59, 61, 63, 78, 79, 89, 90,101, 103 modified differently as compared to mutation (b), 104, 111,118, 130, 133 modified differently as compared to mutation (c), 136,138, 145, 149, 150, 156 modified differently as compared to mutation(d), 157, 161, 164, 167, 173, 181, 183, 184, 185, 191, 197, 199, 204,206, 209, 217 modified differently as compared to mutation (e), 218,224, 228, 234, 235, 242, 243, 245, 247, 248, 249, 251, 252, 253, 259,263, 265, 271, 267, 269, 271, 272, 273 and
 275. 48. The modifiedsubtilisin of claim 43, which further has one or more of the followingmutations: V4A, P5L, Y6N, Y6H, G7S, S9P, S9A, P14T, A15G, A15V, H17L,S18T, Q19H, G20D, S24T, N25D, I31L, S38T, N43S, N43I, N43T, R45S, S49G,F50I, V51I, N56S, Q59L, G61S, S63P, S78P, I79F, S89P, L90I, Y104N,I111V, I111N, N118Y, T130I, T130A, K136N, V138A, S145G, V149I, A150V,E156G, G157D, S161N, T164S, Y167F, N181S, S183N, N184Y, Q185H, S191G,D197G, M199V, S204T, Q206H, Q206L, L209H, N218I, N218T, T224A, A228T,1234N, L235I, L235P, T242A, N243S, N243H, N243T, Q245L, R247H, D248V,R249S, S252G, T253S, N259H, Y263C, Y263N, K265N, L267F, N269H, N269I,N269D, Q271H, Q271L, A272P and Q275L.
 49. The modified subtilisinaccording to claim 1, which has one of the following combinations ofmutations: V51I/Q103R/E156A, Q103R/V138A/A150V/E156A/Y217H/Q271H,S9P/R45S/Q103R/T130A/E156G/S183N/Y217H,G61S/S78P/Q103R/E156A/E54D/I79F/Q103R/E156G/Y217H/N243S,S78P/L90I/Q103R/E156A, Q103K/T130A/E156G/L209H, Q103K/E156A/T242A/N259H,N25D/Q103K/E156A, P14T/A15G/E54D/Q103K/T130A/E156G/S252G/Q271L,Q103K/T133K/N181S/S252G/Q271L, N43I/G61S/S78P/Q103K/T130A/E156G/S204TN43S/E54D/Q103R/S145G/E156K/G157D/M 199V/Y217H/D248V,S18T/N43S/Q103R/V138A/A150V/E156A/Y217H/Q271L, P14T/A15V/H17L/Q103R/T130A/E156A, Q103R/V138A/A150V/E156A/Y217H,Q103R/N181S/S204T/Y217H/T224A/A228T,V4A/E54D/G61S/Q103R/E156A/S183N/Y217H, S9P/E54G/Q103R/I111V/E156A/Y217H,E54D/Q103R/T133K/E156A/Y217H, S24T/G61S/Q103R/E156A/S183N/Y217H/T253S,G20D/N43T/E54D/Q103R/T130A/E156A/S183N/Y217H/L235I,V4A/E54D/G61S/Q103R/S145G/E156A/S183N/Y217H,Q103R/V138A/A150V/E156A/Y217H/D248V/R249S,P14T/A15V/I31L/N43S/E54D/Q103R/E156A,Q19H/I31L/E54D/Q59L/S63P/Q103R/Y104N/T130A/E156G/S161N/S183N/-N243H/L267F/N269H,E54D/Q103R/I111N/N118Y/T130A/E156A/Q185H/I234N/L235P/Q245L/N259H,E54D/Q103R/I111N/N118Y/T130A/E156A/Q185H,E54D/T130A/V149I/E156A/S183N/D197G/Q206H/N218I/N269I,E54D/Q103R/T133K/K136N/E156A/N269D,Y6N/P14T/A15V/I31L/N43S/E54D/Q103R/E156A,P14T/A15V/E54D/Q103R/E156A/Y167F/Y217H/N218T,P14T/A15V/I31L/E54D/Q103R/N118Y/E156A/S191G/N243T/Y263C/N269H,G7S/S9A/Q103R/T133K/E156A/Y217H/T224A/Y263N/A272P,E54D/Q103R/T133K/K136N/E156A/Y217H,S9P/Q103R/T133K/E156A/T164S/S183N/Y217H, E54D/Q103R/T133K/E156A/N184Y/Q206L/Y217H/K265N, P5L/Y6H/S49G/Q103R/T133K/E156A/Y217H,E54G/Q103R/I111V/V138A/A150V/E156A/Y217H/L235I, S89P/Q103R/E156A/Q271H,131L/N43S/N56S/Q103R/V138A/A150V/E156A/Y217H/Q271H,P14T/A15V/E54D/Q103R/E156A/Y217H/Q271H, E54D/Q103R/T133K/E156A,E54D/Q103R/T133K, V4A/E54D/G61S/Q103R/S183N,S9P/E54D/Q103R/I111V/S13N/D248V/R249S andN43S/E54D/Q103R/S145G/E156K/G157D/M199V/Y217H/D248V.
 50. A modifiedsubtilisin comprising the following combination of mutations in an aminoacid sequence of a precursor subtilisin at a position numbered accordingto the amino acid sequence of the mature subtilisin BPN′ shown in SEQ IDNO:1: Y6N/I31L/S38T/F50I/Q103R, P14T/Q103R/T130I/Q185H/K265N, andS145G/E156K/R247H.
 51. The modified subtilisin according to claim 1 or50, wherein the precursor subtilisin is subtilisin E (Bacillus subtilisstrain 168; SEQ ID NO:2), subtilisin BPN′ (Bacillus amyloliquefaciens;SEQ ID NO:1), subtilisin DY (Bacillus subtilis strain DY; SEQ ID NO:3),subtilisin Carlsberg (Bacillus licheniformis; SEQ ID NO:4), subtilisinsavinase (Bacillus lentus; SEQ ID NO:5), or a fragment or derivativethereof, or is a recombinant or engineered subtilisin.
 52. The modifiedsubtilisins according to claim 51, wherein the precursor subtilisin issubtilisin E (SEQ ID NO:2) or a fragment or derivative thereof.
 53. Themodified subtilisin according to claim 52 which has the sequence shownin SEQ ID NOs:10 to
 59. 54. A DNA coding for the modified subtilisin ofclaim
 1. 55. A vector comprising the DNA of claim
 54. 56. Amicroorganism which comprises the DNA of claim
 54. 57. A microorganismwhich comprises the vector of claim
 55. 58. A method for producing amodified subtilisin, which comprises culturing the microorganism ofclaim
 56. 59. A method for producing a modified subtilisin, whichcomprises culturing the microorganism of claim
 57. 60. A detergentcomposition comprising a modified subtilisin of claim
 1. 61. A methodfor degrading proteinaceous material, which comprises treating theproteinaceous material with a modified subtilisin of claim 1.